Protoplasma, Vol. 190, No. 3-4, pp.141-150, 1996

N. A. Durso, J. D. Leslie, and R. J. Cyr

In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1-alpha is associated with microtubules in carrot cells
Figures

Figure 1. Immunoblotting characterization of anti-pp50 antibodies.

Figure 2. Failure of affinity purified anti-pp50 antibodies to recognize in vitro-assembled and taxol-stabilized MTs of pure carrot tubulin.

Figure 3. Co-localization of affinity purified anti-pp50 antibodies (APE50) and anti-tubulin antibodies (tubulin) following treatments with microtubule drugs.

Figure 4. Improved immunolocalization using affinity purified anti-pp50 antibodies.

Figure 5. Co-localization of affinity purified anti-pp50 antibodies (APE50) and anti-tubulin antibodies (tubulin) in a cytokinetic microtubule array.

Figure 6. Co-localization of affinity purified anti-pp50 and anti-tubulin antibodies in a carrot protoplast fixed prior to detergent extraction and examined by a fluorescence resonance energy transfer (FRET) technique.

© 1996 Springer-Verlag. Reproduced with permission.
(from Durso et al. 1996, Protoplasma 190: 141-150)

Abstract
Evidence indicates that elongation factor-1a (EF-1a), a ubiquitous and abundant protein factor involved in the first step of peptide elongation, is also associated with the cytoskeleton in a variety of organisms. Although the effects of these associations on EF-1a`s translational function have not been examined, the associations do appear to result in non-passive effects on the cytoskeleton. A carrot homolog of EF-1-alpha , pp50, has been reported to interact with microtubules in vitro, inducing the formation of microtubule bundles that can be dissociated by calcium/calmodulin. The characterization of anti-pp50 antibodies is reported here. Immunocytochemistry, using anti-pp50 and anti-tubulin antibodies, was used to investigate the co-localization of pp50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp50 appears to be associated with microtubules. Treatment of such protoplasts with amiprophos-methyl (APM) reduced both the presence of microtubules and the co-localizing pp50-associated fluorescence. In taxol-treated protoplasts, increases in both microtubules and the co-localizing pp50-associated fluorescence were observed. When carrot protoplasts were fixed prior to detergent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence resonance energy transfer (FRET) between fluorochromes associated with anti-pp50 and anti-tubulin reporters was observed, indicating that some pp50 is intimately associated with microtubules. The in situ cytoarchitectural evidence is consistent with a function previously proposed for pp50 based on in vitro experiments - that pp50 is a plant microtubule-associated protein (MAP) whose function can be modulated by a calcium/calmodulin signal transduction mechanism in plant cells.

Keywords: Calcium; Calmodulin; Carrot cells; Elongation factor-1-alpha; Immunofluorescence microscopy; Microtubule-associated protein.
Abbreviations: APM amiprophos-methyl; BSA bovine serum albumin; EF-1a elongation factor-1-alpha; FRET fluorescence resonance energy transfer; MAP microtubule-associated protein; PBS phosphate buffered saline; SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis.


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